1. Field of the Invention
The present invention relates generally to mass spectrometry and more specifically to analysis of enzyme-catalyzed chemical reactions, pathways, and reaction mechanisms by quenched-flow mass spectrometry which allows chemical and biochemical reactions to be analyzed at different stages throughout the reaction.
2. Discussion of the Prior Art
Until recently, it was not possible to analyze enzymes or enzyme solutions using mass spectrometry. No method was available to introduce a nonvolatile protein molecule into the gas phase, as is necessary for mass spectral analysis. If a protein molecule dissolved in water was introduced into a mass spectrometer the water would short out and destroy the ionizing filament.
With the recent advent of techniques such as fast atom bombardment (FAB), matrix assisted laser desorption ionization (MALDI), and electrospray ionization (ESI), it now is possible to transfer nonvolatile, ionized protein molecules into the gas phase. The recently developed techniques of flow FAB and electrospray ionization additionally have the advantage of permitting introduction of solutions into the mass spectrometer, thus enabling mass spectral analysis of dissolved enzymes. The gentle ionization conditions of electrospray ionization mass spectrometry make it particularly advantageous for analysis of enzyme reaction mixtures, because covalent and noncovalent enzyme ligand interactions often remain intact. However, the mere fact that the electrospray ionization inlet exists doesn""t provide a method for analyzing chemical and biochemical reactions.
Accordingly, there is a clearly felt need in the art for enzymatic reaction mechanisms by quenched-flow mass spectrometry which allows monitoring the progress of chemical, biochemical and enzymatic reactions at a particular time during the reaction.
The primary objective of the present invention is to provide enzymatic reaction mechanisms by quenched-flow mass spectrometry which allows monitoring the progress of chemical, biochemical and enzymatic reactions at a particular time during the reaction.
According to the present invention, there are several preferred methods for analyzing enzymes with an electrospray mass spectrometer. The hardware required for analyzing enzyme reactions includes, a mass spectrometer, an electrospray ionization inlet, at least two syringes, at least one pushing ram, at least one mixing device, and monitoring equipment. The pushing ram includes a platen for pushing the plunger of at least one syringe. The platen is precisely actuated preferably by a stepper motor, but could be actuated by a DC motor with optical encoder, a motor driven clutch mechanism, a hydraulic system, or the like. The rotation of the stepper motor is preferably microprocessor controlled. The output of each syringe is connected to the mixing device which mixes the reactant contained in each syringe and initiates a chemical reaction immediately, or immediately subsequent to the point of mixing.
The output of the mixing device is connected to the electrospray ionization inlet with a reaction tube having a specific volume. The output of the electrospray ionization inlet is connected to the inlet of the mass spectrometer. The mixed reaction solution is input through the electrospray ionization inlet. The volatile molecules of the solution are removed in a chamber within the electrospray ionization inlet filled with a warm flowing drying gas. The ions generated by the electrospray ionization inlet are transferred through a second orifice or skimmer into the vacuum of the mass spectrometer where they are subjected to mass analysis. The electrospray mass spectrometer is adjusted to collect the ions of interest, and the resulting current is then recorded with the monitoring equipment. A spectrophotometer may also be used for analyzing the mixed solution.
Accordingly, it is an object of the present invention to provide enzymatic and nonenzymatic reaction mechanisms by quenched-flow mass spectrometry which allows the reaction to be stopped at a particular time during the reaction.
It is a further object of the present invention to provide enzymatic and nonenzymatic reaction mechanisms by quenched-flow mass spectrometry which utilizes an electrospray mass spectrometer to analyze an intermediate sample produced at some time during the reaction.
It is yet a further object of the present invention to analyze, obtain, and to quantify a sufficient number of mass analyses at a sufficient number of particular times during a reaction to enable construction of progress curves for every charged, nonvolatile reaction species of unique mass/charge ratio and interest.
Finally, it is another object of the present invention to provide enzymatic and nonenzymatic reaction mechanisms by quenched-flow mass spectrometry which allows a user to combine two existing pieces of equipment to obtain an intermediate sample which was not possible with prior art methods.
These and additional objects, advantages, features and benefits of the present invention will become apparent from the following specification.